核转录因子κB在大骨节病患者中的表达及在软骨细胞凋亡中的作用

The expression of nuclear factor κB signal molecule in Kashin-Beck disease and its role in chondrocyte apoptosis

目的通过观察核转录因子κB（NF-κB）p65在大骨节病患者全血和软骨细胞（C28/I2）中的表达，分析NF-κB p65分子在软骨细胞凋亡中的作用，阐明NF-κB信号通路在大骨节病发病机制中的作用。方法采用病例-对照研究方法，从陕西省大骨节病病区旬邑、永寿、长武、麟游、千阳、陇县选择161例大骨节病患者作为病例组，同时选取性别、年龄相匹配的312名健康人作为对照组，采集病例组和对照组人群肘静脉血，乙二胺四乙酸（EDTA）抗凝，用于NF-κB p65蛋白测定。采用成组设计及细胞培养方法，建立人C28/I2软骨细胞氧化损伤模型，实验分为4组：对照组、叔丁基过氧化氢（tBHP）损伤组（tBHP 300.00 μmol/L）、低硒预保护组[亚硒酸钠（Na2SeO3）0.05 mg/L ＋ tBHP 300.00 μmol/L]、中硒预保护组（Na2SeO3 0.10 mg/L ＋ tBHP 300.00 μmol/L），Hoechst 33342染色检测各组细胞凋亡情况，二氯荧光黄法检测细胞内活性氧（ROS）含量。应用Trizol法从全血和软骨细胞中提取蛋白，采用蛋白质免疫印迹法检测全血及软骨细胞中NF-κB p65信号分子的蛋白表达。结果病例组与对照组在年龄和性别分布上差异均无统计学意义（t= 0.336，P 〉 0.05；χ2= 0.407，P 〉 0.05）。病例组全血中NF-κB p65蛋白表达明显升高，约为对照组的1.835倍（病例组：0.167 ± 0.026，对照组：0.091 ± 0.014；t = 5.147，P 〈 0.01）。荧光显微镜下，tBHP损伤组细胞多呈强蓝色荧光，凋亡细胞数量多；且细胞内ROS含量（1.219 ± 0.104）高于低、中硒预保护组（0.832 ± 0.077、0.635 ± 0.070，P均〈 0.05）。tBHP损伤组NF-κB p65蛋白表达（1.563 ± 0.351）明显高于对照组（0.451 ± 0.069，P 〈 0.05）；低、中硒预保护组NF-κB p65蛋白表达有低于tBHP损伤组的趋势。结论大骨节病患者NF-κB信号通路表达上调，NF-κB p65信号分子在氧化损伤所致的软骨细胞凋亡中具有一定作用，提示NF-κB信号通路在大骨节病发病机制中发挥了一定的作用。

ObjectiveTo clarify the role of nuclear factor κB （NF-κB） signaling pathway in pathogenesis of Kashin-Beck disease （KBD） by observing the expression of NF-κB p65 in the whole blood samples of patients with KBD and controls, and the expression of NF-κB p65 in C28/I2 chondrocyte, and to analyze the role of NF-κB p65 molecule in chondrocyte apoptosis.MethodsThrough a case-control study, 161 patients with KBD （KBD group） were selected from Xunyi, Yongshou, Changwu, Linyou, Qianyang and Long counties in KBD endemic areas and 312 healthy people （control group） were matched by age and sex in Shaanxi Province. Venous blood samples were collected from patients and healthy controls, which were anticoagulated and used for determination of NF-κB p65 protein. According to the group design, the model of C28/I2 chondrocyte oxidative damage was established. The experiments were divided into 4 groups including control group （C）, tBHP injury group （O, tBHP 300.00 μmol/L）, low selenium pre-protection group （OS1, 0.05 mg/L Na2SeO3 ＋ 300.00 μmol/L tBHP）, and middle selenium pre- protection group （OS2, 0.10 mg/L Na2SeO3 ＋ 300.00 μmol/L tBHP）. Then, cell apoptosis was detected by Hoechst 33342 and reactive oxygen species （ROS） was detected by dichlorfluorescein （DCF） method. The protein was extracted by Trizol method, then protein expression level of NF-κB p65 molecule was detected by Western blotting in whole blood samples and C28/I2 chondrocyte.ResultsThe differences in age and sex were not statistically significant between KBD group and control group （t = 0.336, P 〉 0.05; χ2 = 0.407, P 〉 0.05）. The protein expression level of NF-κB p65 in KBD group was 1.835 times as high as that of control group （KBD： 0.167 ± 0.026, control： 0.091 ± 0.014, t = 5.147, P 〈 0.01）. Under the fluorescence microscope, chondrocyte showed strong blue fluorescence in tBHP group and the level of ROS （1.219 ± 0.104） was higher than those of low and middle selenium pre-protection groups （0.832 ± 0.077, 0.635 ± 0.070, P 〈 0.05）. The protein expression level of NF-κB p65 in tBHP group （1.563 ± 0.351） was higher than that of control group （0.451 ± 0.069, P 〈 0.05）, and protein levels of NF-κB p65 had a decreasing tendency in low and middle selenium pre-protection groups compared to tBHP group.ConclusionThe NF-κB signaling pathway is up-regulated in KBD patients, moreover, chondrocyte experiments show that cell apoptosis is mediated via upregulation of NF-κB p65, which suggests NF-κB signaling pathway may play an important role in pathogenesis of KBD.